Much of a bacterium's regulatory potential is included in 5′-untranslated regions (5′-UTRs), which control the expression of physically adjacent downstream genes. Because of this cis action, the regulatory effects of 5′-UTRs are naturally constrained to the attached genes. As a result, 5′-UTRs have become attractive platforms for the parts-based approach to synthetic regulation, in which engineered regulatory parts that sense custom inputs and change the expression of desired genes in response are integrated.
In bacteria, there are two primary types of regulators found in 5′-UTRs that can serve as starting points for designing new parts. The first type of regulators found in 5′-UTRs includes regulators of translation (i.e., cis-regulators of translation), which link inputs to the accessibility of ribosome binding sites (RBSs). From a parts perspective, such regulators are attractive not only because they are well-represented in nature (e.g. riboswitches that sense small molecules, antisense RNA repressors and activators, and RBSs responsive to proteins, nutrients, pH, and temperature), but also because RBS-based interactions can be tuned or designed de novo using increasingly predictive thermodynamic models. However, cis-regulators of translation have a number of basic limitations: they can only control protein synthesis and not the production of RNAs, they are constrained to act on single genes, and they cannot simply be linked together into complex regulatory nodes as initiation at RBSs is a distributive process.
The second type of regulators found in 5′-UTRs includes regulators of transcriptional elongation (i.e., transcriptional continuation), which link cellular inputs to the continuation of RNA polymerase during RNA synthesis. In contrast to regulators of translation, these can control both the production of coding and non-coding RNAs and can act on entire operons including multiple genes. Furthermore, they are inherently composable because when multiple regulators of transcriptional elongation are linked in tandem in a 5′-UTR, the synthesis of the Nth regulator is gated by the decision of the (N−1)th regulator; this predictably yields logic and higher-order functions. Hence, when engineering custom regulators for 5′-UTRs, one faces a restrictive tradeoff between ease of design for cis-regulators of translation and versatility of output for cis-regulators of transcriptional continuation. Thus, there is a need for custom regulators that are easy to design, that operate on entire operons and that can be composed into logics and higher-order functions. However, such regulators are difficult to engineer because their mechanisms involve action on a moving RNA polymerase, requiring the consideration of poorly defined kinetic and dynamic structural factors in their design. The existence of only a handful of synthetic regulators of transcriptional elongation, based either on riboswitches or the pT181 system, testifies to this difficulty.